As molecular biological techniques are advancing, various genetic engineering methods have been developed in order to produce useful metabolic substance and to modify the substance in an effective form. In practice, a lot of studies on plant metabolic engineering and plant molecular farming have been accomplished. The plant metabolic engineering is to utilize a plant for mediator and the plant molecular farming is to produce high value-added non-plant edible vaccines from non-plant and recombinant proteins for medical use etc from plant. However, there are various technical difficulties and research outcomes are not successful yet.
Especially in order to regulate a metabolic process, it may be useful to express several genes coincidently under a condition. Presently, model researches using reporter genes in tobacco were just attempted in this field. In contrast to prokaryotes, it is difficult to regulate multi-cistronic genes expression in eukaryotes such as plant and animal. The eukaryote has a feature to express only one gene from one promoter by a mono-cistronic mRNA mechanism.
In general, Agrobacterium is applied to transform a plant by inserting a T-DNA. In this case, (1) introduction of multi-cassettes in one T-DNA; (2) co-transformation of Agrobacterium; (3) sexual crossing between transformants; and (4) insertion of multi-cistronic genes into a cassette; are often conducted in order to introduce and express multi-cistronic genes coincidently.
Precisely, the method for introducing the multi-cassettes in one T-DNA is popularly used. Unfortunately, it is hard to express more than two target genes excluding a selective marker gene with one T-DNA vector, because the total number of cassettes for transformation is limited to 3 or 4. Furthermore, when conducting the Agrobacterium mediated co-transformation, each T-DNA should have a different selective marker in order to select transformants. In this case, the selective marker may be a limiting factor to block the plant transformation. Therefore, this procedure is disadvantageous to restrict applicable fields.
The method for stacking genes by using a sexual crossing between transformants collects useful traits of each individual into a plant. But, this procedure needs to consume a long time period and is hard to control a result exactly.
Besides, the method for introducing multi-cistronic genes into a cassette has been tried to precede metabolic engineering studies and to obtain complex resistance traits against stress or diseases. In this process, the eukaryotic mechanism of protein expressions is applied so that specific recognition sites of a eukaryote are introduced at between target genes.